Activating K-RAS mutations occur in >95% of patients with advanced pancreatic cancer, and recent studies identify CRAF as a kinase required for tumorigenesis downstream of oncogenic K-RAS. Although CRAF kinase activity is traditionally linked to tumor progression via activation of MAPK signaling, we have focused our attention on two non-canonical CRAF pathways which are independent of CRAF catalytic activity and activation of MAPK. Several years ago, we established that CRAF phosphorylation on serine 338 (pS338) drives CRAF translocation to the mitochondria where it forms a complex with and inactivates the pro-apoptotic kinase ASK1 to promote cell survival. In a new manuscript in press at Nature Medicine, we now report that CRAF pS338 also forms a complex with the cell cycle kinase Plk1 at the mitotic spindle to impact cell cycle progression. Accordingly, genetic or allosteric inhibition of this serine on CRAF drives both apoptosis and mitotic arrest that is independent of the MAPK pathway and not observed with inhibitors of MAPK signaling. Our overall objective is therefore to target CRAF function and exploit its role in both cell survival ad mitotic progression, in order to develop novel therapeutic approaches for pancreatic cancer patients. The proposed studies will define how these two unique MAPK-independent functions of CRAF contribute to progression and metastasis of pancreatic cancer driven by oncogenic K-RAS. In Aim 1 we determine the mechanisms by which the non-canonical CRAF pS338 pathway is activated in the unique genetic context of pancreatic cancer. Specifically, we will focus on how both K-RAS and PAK regulate CRAF pS338, since these molecules are known to be dysregulated in pancreatic cancer. The goal of Aim 2 is to evaluate the contributions of the two non-canonical CRAF pS338 pathways on pancreatic cancer cell survival (via ASK1) and cell proliferation (via Plk1) as these relate to tumor growth and metastasis of pancreatic cancer in vivo. Since mitosis is the most radiosensitive stage of the cell cycle, the goal of Aim 3 is to use mouse models of pancreatic cancer and metastasis to understand how to optimally combine ionizing radiation with blockade of this novel CRAF pS338 pathway which induces mitotic arrest. We anticipate that our innovative approach to target tumor cell survival, proliferation, and radiosensitivity via the K-RAS/CRAF pathway will lead to exciting new opportunities for metastatic pancreatic cancer. PUBLIC HEALTH RELEVANCE: The vast majority of pancreatic cancers express oncogenic K-RAS, which is well known to trigger a number of canonical signaling pathways promoting tumor progression and metastasis. In contrast, our project focuses on defining a novel non-canonical K-RAS driven pathway by which CRAF phosphorylation on serine 338 drives tumor cell cycle progression and cell survival. Since cells undergoing mitosis are the most sensitive to the DNA-damaging effects of radiation, we propose that perturbing this K-RAS/CRAF axis will provide a novel means to improve the response to ionizing radiation that is a standard component of multimodal therapy for patients with metastatic pancreatic cancer.